Method for crosslinking of haemoglobin

ABSTRACT

Haemoglobin is crosslinked by means of transglutaminase, whereafter the transglutaminase is inactivated. The crosslinked haemoglobin can be used as a molecular agent for oxygen transportation, as an oxygen scavenger, and as a constituent of a food composition.

The invention comprises a method for crosslinking of haemoglobin and ause of such crosslinked haemoglobin.

From Biotechnology of Blood, edited by Jack Goldstein, 1991, page 108,first full paragraph it appears that the prior art methods forpolymerisation of haemoglobin are associated with several drawbacks,especially the heterogenous distribution of polymers due to theunspecificity of the hitherto used crosslinking agents and the fact thatthe degree of polymerisation is difficult to control.

Thus, it is the purpose of the invention to provide a method forcrosslinking of haemoglobin, in regard to which the crosslinkedhaemoglobin exhibits a more homogenous distribution of polymers and abetter controllable degree of polymer, and a use of such crosslinkedhaemoglobin.

The method according to the invention for crosslinking of haemoglobin ischaracterized by the fact that haemoglobin present in an aqueoussolution thereof with a pH between 5 and 9 is crosslinked by means oftransglutaminase (EC 2.3.2.13), whereafter the crosslinking is stoppedat the wanted degree of polymerisation.

Surprisingly it has been found that transglutaminase is a very specificcrosslinking agent, and that it is possible to obtain a very goodcontrollable degree of polymerisation by means of the method accordingto the invention.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the pH value of the aqueous solution isbetween 6.5 and 8. The crosslinking reaction proceeds with a mostsatisfactory velocity in this pH interval.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the maximum amount of transglutaminase is2% of pure transglutaminase protein, calculated in regard to thehaemoglobin protein. Higher levels of transglutaminase than 2% willprove to be uneconomic. Even at very low levels below 2% thecrosslinking will proceed, if given the time.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the crosslinking is stopped by heatinactivation of the transglutaminase, preferably by heating to up to 65°C. for 5 minutes. This is the most simple and effective way ofinactivation.

Also, the invention comprises a use of the crosslinked haemoglobinproducible by means of the method according to the invention, due to anyof the inherent properties of the crosslinked haemoglobin.

A preferred embodiment of the use of the crosslinked haemoglobinproducible by means of the method according to the invention is as amolecular agent for oxygen transportation, i.e. as a blood substitute.The desirability of new blood substitutes are well known, videBiotechnology of Blood, edited by Jack Goldstein, 1991, page 101. It hasbeen found that it is easy to produce the crosslinked haemoglobinaccording to the invention with a molecular weight well above 64,000Dalton, which is above the cut-off value of the kidneys, 60,000 Dalton.This is one of the conditions for a satisfactory blood substitute.

A preferred embodiment of the use of the crosslinked haemoglobinproducible by means of the method according to the invention is as anoxygen scavenger. It has been found that this haemoglobin functionseffectively as an oxygen scavenger, e.g. in food items, and thataddition of antioxidants hereby can be eliminated or reduced.

A preferred embodiment of the use of the crosslinked haemoglobinproducible by means of the method according to the invention is as aconstituent of a food composition.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 demonstrates the increase in viscosity with time for thepolymerization of haemoglobin with transglutaminase.

EXAMPLE 1

To 200 g of a 14% solution (w/w) of haemoglobin (Merck, Darmstadt, BRD)(pH 7.0, temperature 37° C.) with a concentration of Ca⁺⁺ of 5 mMtransglutaminase was added in an amount of 0.4% (w/w) in relation to theamount of haemoglobin, i.e. 112 mg. The transglutaminase was produced asindicated in Example 1 of WO 93/15234. The reaction was carried out for200 minutes, and subsequently the activity of the transglutaminase wasinactivated by heating to 65° C. for 5 minutes. Reference is made toFIG. 1, which shows the progression of the polymerization expressed asviscosity. The crosslinked haemoglobin was isolated by lyophilization.

I claim:
 1. A method for crosslinking a hemoglobin, comprising(a)treating an aqueous solution which comprises the hemoglobin with atransglutaminase wherein the aqueous solution has a pH between 5 and 9;and (b) inactivating the transglutaminase at a desired degree ofpolymerization.
 2. The method according to claim 1, wherein the aqueoussolution has a pH between 6.5 and
 8. 3. The method according to claim 1,wherein the maximum amount of the transglutaminase is 2% of puretransglutaminase protein, calculated in regard to the haemoglobinprotein.
 4. The method according to claim 1, wherein thetransglutaminase is inactivated by heating to up to 65° C. for 5minutes.